Two step enrichment facilitates background reduction for proteomic analysis of lysosomes

ABSTRACT: Lysosomes comprise the main degradative compartment of almost all mammalian cells and are involved in various cellular functions, most of which are catalyzed by the lysosomal proteome. Lysosomal proteins are low abundant, complicating their analysis by mass spectrometry-based proteomics. To increase analytical performance, and to enable profiling of lysosomal content, lysosomes are frequently enriched, for which currently two approaches are most commonly used: superparamagnetic nanoparticles (SPIONs) and immunoprecipitation from cells overexpressing a 3xHA-tagged version of TMEM192 (TMEM-IP). We investigated the effects of both approaches on the lysosomal proteome through enrichment of lysosomes with the respective other approach proteomic analyses. For SPIONs treatment, we identified mainly an effect on cellular iron homeostasis, while changes of the lysosomal proteome were moderate. For overexpression of TMEM192, we observed more pronounced effects in lysosomal protein expression, especially related to alterations of membrane proteins and such involved in protein trafficking. Furthermore, we established a combined strategy consisting of sequential lysosome enrichment with SPIONs and TMEM-IPs. This approach enabled increased purity of lysosome enriched fractions and, through TMEM-IP-based lysosome enrichment from SPIONs flow through and eluate fractions, further characterization of the individual approaches’ properties.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Sara Bonini  

LAB HEAD: Dominic Winter

PROVIDER: PXD048696 | Pride | 2024-08-01

REPOSITORIES: Pride